A method for preparing adjustable functional black plum extract and application thereof

Abstract

The application discloses a kind of function adjustable dark plum black essence directional preparation method and its application.The application uses dark plum as raw material, through hot air drying, conventional smoking, three progressive heat processing driven Maillard reaction gradient of depth smoking, obtain the dark plum precursor of different Maillard reaction degree;Again through degreasing, ultrasonic-assisted extraction, reduced pressure concentration, alcohol precipitation, ultrafiltration membrane purification, freeze-drying, obtain high-purity dark plum black essence.The application is prepared two kinds of function differentiation black essence by regulating processing intensity: conventional smoking dark plum black essence has the best amphiphilic, can form dense elastic interface film and three-dimensional gel network, as natural emulsifying stabilizer, its 21 days emulsion stability is better than gelatin and coffee black essence;Depth smoking dark plum black essence has highly crosslinked structure, can realize polyphenol colon target slow release.The multifunctional black essence provided by the application can be widely applied to food emulsion, functional food and active ingredient delivery system.

Description

Technical Field

[0001] This invention relates to the field of bioproduct processing and preparation technology, specifically to a method for the directional preparation of melanin-like substances from plums based on gradient Maillard reaction regulation, and the application of the prepared melanin-like substances in food emulsifying stabilizers and bioactive carriers. Background Technology

[0002] Melanoidins are nitrogen-containing, brown macromolecular polymers formed in heat-processed foods through the final stage of the Maillard reaction between reducing sugars and amino compounds (amino acids, peptides, or proteins). They are widely found in everyday foods such as coffee, baked goods, cocoa, and beer. It is estimated that humans ingest 10–12 g of melanoidins daily from their diet, and they hold significant potential as natural bioactive components in the development of functional foods.

[0003] Currently, research on melanoidins mainly focuses on traditional heat-processed foods such as coffee, vinegar, and bread. Coffee melanoidins have been proven to possess various physiological activities, including antioxidant, antibacterial, and prebiotic properties. Their structure is primarily a protein-polysaccharide complex, and they can be separated and purified using methods such as ultrafiltration (with a molecular weight cutoff of 10 kDa). Existing melanoidin preparation technologies mostly employ alkaline solution extraction combined with acid precipitation or organic phase separation. For example, EP4108094A1 discloses a method for preparing melanoidin products from coffee grounds, which involves extraction with an extractant at pH > 7, followed by acid precipitation or organic phase separation to obtain melanoidin precipitate, which can then be further fractionated by molecular weight through ultrafiltration. Furthermore, in fermented foods such as soy sauce and vinegar, melanoidin extraction also primarily employs methods such as ultrafiltration, macroporous resin adsorption, and dextran gel chromatography.

[0004] In recent years, research on melanoidins in processed fruit and vegetable products has received increased attention. Studies show that the melanoidins in dried apple slices are polysaccharide-type melanoidins, exhibiting blue-green fluorescence, and different drying methods (hot air drying, pressure difference flash drying) can affect their molecular weight and conjugated system. However, for dried plums (which are mainly composed of polysaccharides, pectin, and organic acids), research has become less focused on melanoidins. Prunus mume The formation patterns and functional regulation of melanoidins in the matrices of specialty fruits such as _____ still lack systematic research.

[0005] Current technology still has the following significant shortcomings: First, melanin from *Prunus mume* has not yet been developed. *Prunus mume* is a plant belonging to the Rosaceae family, specifically the plum (*Prunus mume*). Prunus mumeThe dried, nearly mature fruit of the plum (Prunus mume) is rich in polysaccharides, pectin, organic acids, and various amino acids, making it an ideal raw material for researching fruit-derived melanoidins. However, current research on plums mainly focuses on small molecule compounds such as organic acids, flavonoids, and volatile components. There are no reports on the extraction, preparation, or functional applications of high-molecular-weight melanoidins generated by the Maillard reaction during the drying and smoking process of plums. This important component, melanoidins from plums, remains a completely unrecognized and unexplored area in current technology.

[0006] Secondly, the structure-function relationship of fruit-derived melanoidins has not yet been established, resulting in poor functional tunability. Existing research on melanoidins mainly focuses on protein / starch matrix systems such as coffee and cereals, while the formation rules and structural characteristics of fruit-derived melanoidins, whose main structural framework is polysaccharides and pectin, remain unclear. More importantly, current technologies cannot controllably prepare differentiated melanoidins with different molecular weights, degrees of polymerization, and surface properties by regulating processing conditions. This means that the functions of melanoidins (such as emulsification stability, antioxidant properties, and sustained-release delivery) cannot be targeted and adjusted according to application requirements.

[0007] Third, existing preparation methods struggle to achieve gradient control of melanoidin structure and function. Existing melanoidin extraction technologies often employ a combination of extraction with fixed process parameters and ultrafiltration purification, but they cannot obtain a series of melanoidin products with differentiated structures and functions by systematically controlling the Maillard reaction process. For example, conventional ultrafiltration can only achieve molecular weight cutoff, but it is difficult to gradient control key structural parameters such as the accumulation of Maillard reaction intermediates, the degree of aromatic condensation, and surface hydrophobicity. Therefore, it is impossible to achieve functionally targeted design of melanoidins from "antioxidant-dominant" to "emulsion-stabilizing-dominant" and then to "slow-release delivery-dominant."

[0008] Fourth, the application value of melanoidins from dried plums as functionally modifiable food ingredients has not been explored. Dried plums are rich in polysaccharides, pectin, and various amino acids, making them an ideal model for studying the functional modifiability of fruit-derived melanoidins. However, existing technologies have not yet provided any technical insights into how to drive the directional evolution of the Melarad reaction in dried plums through gradient smoking processing to obtain a series of melanoidins with differentiated molecular weights, degrees of polymerization, and surface hydrophilicity-hydrophobicity balance, and to establish a precise matching relationship between "processing parameters-melanoidin structure-functional properties".

[0009] In summary, as a novel natural product component that has not yet been recognized and developed, there is an urgent need to develop a directional preparation method for tunable ume melanin. By systematically controlling the Maillard reaction gradient, the controllable preparation of ume melanin with different functional orientations (emulsion stabilization type, antioxidant type, and sustained-release delivery type) can be achieved, thus filling the gap in the technology for tunable preparation of fruit-derived melanin. Summary of the Invention

[0010] The purpose of this invention is to provide a method for the targeted preparation of melanin-like products with adjustable functions, which can be used to prepare melanin-like products with different properties and functions.

[0011] To achieve the above objectives, the present invention provides a method for the directional preparation of melanin from dried plums with adjustable functions, comprising the following steps: (1) Place fresh dried plums ( Prunus mume The fruit is subjected to gradient processing, wherein the gradient processing is selected from at least one of hot air drying, conventional smoking or deep smoking, to obtain processed plum products with different Maillard reaction degrees. (2) Remove the pits from the processed plums obtained in step (1), crush them, sieve them, defatt them with petroleum ether, add an ethanol solution with a volume concentration of 8%~12%, the material-liquid ratio g:mL is 1:15~1:25, perform ultrasonic-assisted extraction at 70~90℃, the ultrasonic power is 200~400W, the frequency is 30~50kHz, and the extraction time is 20~40min; (3) Centrifuge the extract obtained in step (2), take the supernatant and concentrate it under reduced pressure to 1 / 4 to 1 / 6 of the original volume, add anhydrous ethanol to the final concentration of 70% to 85%, let it stand to precipitate, centrifuge and take the supernatant. (4) The supernatant obtained in step (3) is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The retentate is collected and freeze-dried to obtain crude black plum extract. Further, the hot air drying in step (1) is drying at 80℃ for 72 hours; the conventional smoking is smoking at 60~80℃ for 72 hours; and the deep smoking is smoking at 80~110℃ for 96 hours.

[0012] Further, in step (2), the volume concentration of the ethanol solution is 10%, the material-to-liquid ratio (g:mL) is 1:20, the extraction temperature is 80℃, the ultrasonic power is 300W, the frequency is 40kHz, and the extraction time is 30min.

[0013] A melanin derived from dried plums prepared by the above method, wherein the melanin derived from dried plums has the following characteristics: (i) The total polysaccharide content of the melanin in the plum variety is 418.01±28.13~649.37±12.60 mg / g, the total phenol content is 38.70±1.69~57.44±3.27 mg / g, the protein content is 25.65±3.18~57.05±1.84 mg / g, and the total flavonoid content is 33.37±2.69~48.11±2.67 mg / g; (ii) The molecular weight distribution range of the melanin in the plum-type product is 33.96±0.05 kDa~163.92±0.11 kDa; (iii) The scanning electron microscope image of the melanin-like substance described above shows that it has a loose porous sheet-like structure, a dense blocky structure, or a uniform honeycomb microporous structure; the Fourier transform infrared spectrum shows that it has a structure in the range of 3417~3433 cm⁻¹. -1 It exhibits an OH / NH stretching vibration peak at 1640–1655 cm⁻¹. -1 It exhibits an amide I band and an aromatic C=C stretching vibration peak, in the range of 1000–1250 cm⁻¹. -1 The presence of COC and sugar ring characteristic peaks indicates that the melanin in the plum-type product is rich in hydroxyl, amino, carbonyl, and glycosidic groups.

[0014] Furthermore, the melanin from dried plums is conventional smoked melanin from dried plums (MSW), with a molecular weight of 50.68±0.10 kDa, a total polysaccharide content of 649.37±12.60 mg / g, a total phenol content of 57.44±3.27 mg / g, a total flavonoid content of 48.11±2.67 mg / g, and a contact angle of 40°~60°, exhibiting amphiphilicity.

[0015] Alternatively, the melanin in the plum is deep-smoked melanin in plum (MDSW), with a molecular weight of 163.92±0.11kDa, a protein content of 57.05±1.84 mg / g, a contact angle close to 90°, and hydrophobic and slow-release polyphenol properties.

[0016] Furthermore, the melanin derived from plum possesses antioxidant activity and DPPH free radical scavenging ability (IC). 50 The IC50 value of ABTS free radical scavenging ability ranged from 1420±30.25 to 1762±25.32 μg / mL. 50 The values ​​ranged from 571.56±50.25 to 830.25±61.05 μg / mL.

[0017] Furthermore, the melanin-like substance described herein possesses hypoglycemic activity and α-glucosidase inhibitory activity (IC50). 50 The IC50 value for α-amylase inhibitory activity ranged from 49.5±6.23 to 60.5±3.66 μg / mL. 50 The values ​​ranged from 38.5±3.52 to 60.5±3.66 μg / mL.

[0018] The application of melanin-like substances from plums, as described above, in the preparation of food emulsifying stabilizers.

[0019] Furthermore, the melanin in the plum is conventional smoked melanin (MSW), which can form a stable oil-in-water emulsion. It does not flocculate or aggregate during a 21-day storage period. The emulsion has pseudoplastic fluid properties and an elastic gel network structure, and its storage modulus G′ is higher than its loss modulus G″.

[0020] The application of melanin-like substances from ume as described above in the preparation of colon-targeted delivery vectors.

[0021] Furthermore, the melanin in the plum is deep smoked melanin (MDSW), which has a polyphenol release rate of less than 50% in simulated gastric juice, and achieves sustained release in simulated intestinal and colonic juice, with a total release rate of over 95% within 600 minutes.

[0022] The advantages and positive effects of this invention are as follows: This invention marks the first time that melanoidins have been prepared from dried plums, filling a technological gap in the development of fruit-derived melanoidins. Dried plums are rich in polysaccharides, pectin, organic acids, and various amino acids, making them an ideal raw material for fruit-derived melanoidins. This invention successfully achieved the controllable preparation of dried plum melanoidins through gradient processing.

[0023] This invention achieves systematic control of the Maillard reaction process through gradient processing (hot air drying, conventional smoking, and deep smoking), thereby obtaining a series of plum-derived melanin with differentiated structures, molecular weights, and functional properties. Among them, conventionally smoked plum-derived melanin (MSW) exhibits amphiphilic characteristics (contact angle 40°~60°), a molecular weight of 50.68 kDa, and a total phenol content as high as 57.44 mg / g; deeply smoked plum-derived melanin (MDSW) exhibits hydrophobic characteristics (contact angle close to 90°), a molecular weight as high as 163.92 kDa, and a protein content of 57.05 mg / g.

[0024] The conventional smoked plum melanin (MSW) prepared by this invention exhibits excellent emulsification stability. MSW can rapidly adsorb at the oil-water interface, forming a dense and elastic interfacial film, while simultaneously increasing the viscosity of the continuous phase. The emulsion remains uniform and stable during a 21-day storage period, without flocculation or aggregation. Its emulsification stability is significantly superior to that of coffee melanin and gelatin, making it suitable as a natural, clean-label, plant-based emulsification stabilizer.

[0025] The deep-smoked plum melanin (MDSW) prepared by this invention has a unique colon-targeted delivery function. The highly cross-linked and compact structure of MDSW results in a polyphenol release rate of less than 50% in simulated gastric juice, effectively protecting polyphenols from degradation by gastric acid and digestive enzymes; it achieves sustained release in simulated intestinal and colonic juices, with a total release rate of over 95% within 600 minutes, and can be used as an oral colon-targeted delivery carrier for the development of functional foods.

[0026] The melanin derived from ume prepared by this invention exhibits significant antioxidant activity and a DPPH free radical scavenging capacity of IC50. 50 The concentration range of ABTS is 1420~1762 μg / mL, and its free radical scavenging capacity IC50 is [missing value]. 50The activity ranged from 571.56 to 830.25 μg / mL, and was closely related to the degree of Maillard reaction. Conventionally smoked samples retained more active groups due to moderate polymerization, and their antioxidant activity was superior to that of deeply smoked samples.

[0027] The melanin derived from dried plum prepared in this invention exhibits significant hypoglycemic activity and α-glucosidase inhibitory activity (IC50). 50 The effective concentration ranged from 49.5 to 60.5 μg / mL, and the IC50 for α-amylase inhibitory activity was [missing value]. 50 With a concentration of 38.5~60.5 μg / mL, it can be used as a natural hypoglycemic ingredient for dietary intervention in diabetic patients.

[0028] This invention establishes a precise matching relationship between "processing parameters - melanoidin structure - functional characteristics" for melanoidins derived from plums, enabling the targeted preparation of melanoidins with different functional orientations (emulsion stabilization type, antioxidant type, and sustained-release delivery type), and providing a technical paradigm for the functional tunability development of fruit-derived melanoidins.

[0029] This invention employs an extraction process combining ultrasound-assisted extraction with 10 kDa ultrafiltration membrane retention, resulting in high extraction efficiency and gentle operation, avoiding the damage to the active structure of melanoidins caused by prolonged high-temperature treatment. Optional macroporous resin and dextran gel column purification steps can further improve the purity of melanoidins, meeting the purity requirements of different application scenarios. Attached Figure Description

[0030] Figure 1 Characterization diagrams of MDW, MSW, MDSW and control MC melanin samples prepared in Example 1 of this invention; wherein, Figure 1 In the figure, A is a macroscopic appearance image of the four samples MDW, MSW, MDSW, and MC, and B is a scanning electron microscope morphology observation image of the four samples under progressive magnification conditions of 500x, 2000x, and 10000x.

[0031] Figure 2 These are microscopic characterization images of the storage stability of different types of melanin-stabilized emulsions prepared in Example 1 of this invention; wherein, Figure 2 In the diagram, A represents an emulsion sample without stabilizer, B represents an emulsion sample with 2wt% gelatin as stabilizer, C represents an emulsion sample with 2wt% MDW as stabilizer, D represents an emulsion sample with 2wt% MSW as stabilizer, E represents an emulsion sample with 2wt% MDSW as stabilizer, and F represents an emulsion sample with 2wt% MC as stabilizer. The sub-figures (1), (2), (3), (4), (5), and (6) correspond to the observation results after 0, 3, 5, 7, 14, and 21 days of storage at room temperature, respectively. The scale bar in the lower right corner of each microscopic image is 40μm, and the corresponding macroscopic photograph of the emulsion is embedded in the lower left corner of the image. The magnification of all microscopic images is kept consistent.

[0032] Figure 3 This is a droplet size characteristic diagram of different types of melanin-stabilized emulsions stored at room temperature for 21 days in Example 1 of the present invention; wherein, Figure 3 In the diagram, A represents the droplet size distribution of the emulsion without stabilizer during the storage period; B represents the droplet size distribution of the emulsion with 2 wt% gelatin as stabilizer during the storage period; C represents the droplet size distribution of the emulsion with 2 wt% MDW as stabilizer during the storage period; D represents the droplet size distribution of the emulsion with 2 wt% MSW as stabilizer during the storage period; E represents the droplet size distribution of the emulsion with 2 wt% MDSW as stabilizer during the storage period; F represents the droplet size distribution of the emulsion with 2 wt% MC as stabilizer during the storage period; and G represents a bar chart comparing the average droplet size of the six corresponding emulsions.

[0033] Figure 4 These are rheological property characterization diagrams of different types of melanin-stabilized emulsions in Example 1 of the present invention; wherein, Figure 4 In the diagram, A represents the apparent viscosity of emulsions stabilized with shear rate for those without stabilizer, 2wt% gelatin, 2wt% MDW, 2wt% MSW, 2wt% MDSW, and 2wt% MC, respectively. B, C, D, and E are the storage modulus, loss modulus, complex viscosity, and loss factor curves obtained from frequency scanning tests, respectively. F, G, H, and I are the storage modulus, loss modulus, complex viscosity, and loss factor curves obtained from strain scanning tests, respectively.

[0034] Figure 5 The graph shows the polyphenol release rate changes of MDW, MSW, MDSW and MC prepared in Example 1 of this invention in an in vitro simulated digestion system. The horizontal axis represents the in vitro digestion time, and the vertical axis represents the polyphenol release percentage. The curves correspond to the four groups of samples: MDW, MSW, MDSW and MC. The graph is divided into three digestion stages along the time axis: pH=1.2 simulating a strongly acidic environment of gastric juice, pH=6.8 simulating a weakly acidic environment of intestinal juice, and pH=7.4 simulating a weakly alkaline environment of colonic juice. Schematic diagrams of the corresponding digestive organs are also provided.

[0035] Figure 6 The graph shows the in vitro antioxidant and hypoglycemic enzyme inhibitory activities of MDW, MSW, MDSW, and MC prepared in Example 1 of this invention; wherein, Figure 6In the diagram, A represents the DPPH radical scavenging rate as a function of concentration for each sample and positive control; B represents the ABTS radical scavenging rate as a function of concentration for each sample and positive control; C represents the total reducing power absorbance as a function of concentration for each sample and positive control; D represents the α-glucosidase inhibition rate as a function of concentration for each sample and positive control; and E represents the α-amylase inhibition rate as a function of concentration for each sample and positive control. Positive controls for DPPH, ABTS, and total reducing power experiments were ascorbic acid and water-soluble vitamin E; positive controls for α-glucosidase and α-amylase inhibition experiments were acarbose.

[0036] Figure 7 The graphs show the stability and degradation characteristics of the aqueous solutions of MDW, MSW, MDSW, and MC prepared in Example 1 of this invention; wherein, Figure 7 In the figure, A represents the degradation rate change curve of the four types of melanin aqueous solution stored under ambient sunlight for 52 days, B represents the degradation rate change curve of the four types of melanin aqueous solution stored under 6W, 365nm ultraviolet light irradiation for 15 days, and C represents the bar chart comparing the degradation rates of the four types of melanin aqueous solution placed under different pH environments for 7 days; all degradation rates are calculated based on absorbance at 280nm. Detailed Implementation

[0037] This patent application was supported by the National Key Research and Development Program of China (Research and Application Demonstration of Key Technologies for Characteristic Chinese Medicinal Herbs Industry, No.: 2023YFD1600400).

[0038] This study aimed to elucidate the structure, physicochemical properties, and emulsifying performance of plum-derived melanin processed under gradient smoking intensities, and to compare it with coffee-derived melanin. First, the chemical composition, molecular weight distribution, structural characteristics, thermal properties, and surface wettability of plum-derived melanin were systematically analyzed. Based on this, their in vitro antioxidant and hypoglycemic activities, as well as the bioavailability of polyphenols, were evaluated. Furthermore, the emulsifying stability of these melanin-derived melanin in oil-in-water emulsions was assessed using droplet size distribution, optical microscopy, and rheological measurements. This study is expected to provide valuable reference for the development and utilization of polysaccharide-based fruit-derived melanin as natural, clean-label emulsifiers and bioactive ingredients in functional food applications.

[0039] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0040] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0041] Example 1: Preparation of different functional types of melanin from dried plum 1. Preparation of hot-air dried ume melanin (MDW) Fresh dried plums (from Leshan, Sichuan, 30±5mm in diameter) were collected and evenly spread on trays (3-5cm thick) in a forced air drying oven. The plums were continuously dried with hot air at 80℃ and an airflow velocity of 1.5m / s for 72 hours. Temperature and airflow velocity were recorded every 12 hours. After cooling to room temperature, the moisture content (wet basis) was measured to be ≤5%, yielding the hot-air-dried plum pulp.

[0042] Take 100g of the above-mentioned hot-air dried plum pulp, pulverize it, and pass it through an 80-mesh sieve. Defatt it twice with petroleum ether at room temperature, 2 hours each time. After defatting, place it in a cool, ventilated place for at least 24 hours to completely remove residual organic solvents. Add a 10% (v / v, dry weight) ethanol solution at a material-to-liquid ratio of 1:20, and perform ultrasonic-assisted extraction for 30 minutes at an ultrasonic temperature of 80℃, ultrasonic power of 300W, and frequency of 40kHz. After extraction, centrifuge the mixture at 6000×g for 30 minutes, discard the insoluble residue, and collect the supernatant. Place the supernatant in a rotary evaporator and concentrate it under reduced pressure to 1 / 5 of its original volume at 60℃ and a vacuum of 50-70 mbar. Under continuous stirring, slowly add anhydrous ethanol to the concentrate to a final concentration of 80%, and let it stand for 12 hours to precipitate polysaccharides and other macromolecular impurities. Centrifuge again (6000×g, 30 min), collect the supernatant, and perform cyclic ultrafiltration three times using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove impurities such as small molecule pigments, monosaccharides, and organic acids. The purified melanoidin solution was freeze-dried at -55℃ and 10 Pa vacuum for 48 h to obtain melanoidin with low Maillard reaction degree (MDW). The product was a brownish-yellow powder with a yield of 15.93±1.53%.

[0043] 2. Preparation of conventional smoked plum melanin (MSW) Fresh plum fruits were taken and spread evenly in a thickness of 3-4 cm. They were then smoked with smoldering oak chips at 60-80℃ for 72 hours, stirring once per hour to ensure uniform smoking. After smoking, the samples were air-dried at 30-34℃ for 2-3 days until the moisture content was below 5%, yielding plum raw materials with a moderate degree of Maillard reaction.

[0044] Subsequently, the same method as in '1' above was used for pulverization, sieving, defatting, ultrasonic extraction, centrifugation, concentration, alcohol precipitation, ultrafiltration, and freeze drying to obtain conventional smoked plum melanin (MSW). The product was a dark brown powder with a yield of 20.14±1.25%.

[0045] 3. Preparation of Deeply Smoked Plum Melanin (MDSW) Fresh plum fruits were taken and spread evenly in a thickness of 3-4 cm. They were then smoked at 80-110℃ for 96 hours, stirring once every hour to ensure uniform smoking. After smoking, the samples were air-dried at 30-34℃ for 2-3 days until the moisture content was below 3%, yielding plum raw materials with a high degree of Maillard reaction.

[0046] Subsequently, the product was pulverized, sieved, defatted, ultrasonically extracted, centrifuged, concentrated, precipitated with alcohol, ultrafiltered, and freeze-dried using the same method as described in '1' above, to obtain Deep Smoked Plum Melanin (MDSW). The product was a dark brown powder with a yield of 24.87±1.25%.

[0047] 4. Preparation of coffee melanin (MC) control samples Take 100g of dark roast coffee powder (originating from Azerbaijan, roasted at 180-200℃ for 15-20min), and perform petroleum ether defatting, 10% ethanol ultrasonic extraction, centrifugation, vacuum concentration, 80% ethanol precipitation, 10kDa ultrafiltration purification, and freeze drying to obtain coffee melanin (MC). The product is a dark reddish-brown powder, which will be used for subsequent performance comparison.

[0048] Example 2: Physicochemical properties and structural characterization of melanin from dried plum The dried plum melanin (MDW), conventionally smoked plum melanin (MSW), deeply smoked plum melanin (MDSW), and coffee melanin reference standard (MC) prepared in Example 1 were systematically characterized.

[0049] 1. Chemical composition and yield Total polysaccharide content was determined using the phenol-sulfuric acid method, pectin content using the carbazole method, protein content using the Bradford method, free amino acid content using the ninhydrin method, total polyphenol content using the Folin-Ciocalteu method, total flavonoid content using the aluminum trichloride method, and ash content using the high-temperature ignition method. The results are shown in Table 1.

[0050] Table 1. Chemical composition and yield of different types of melanin The results showed that with increasing fumigation intensity, the yield of melanin-like compounds from dried plums gradually increased from 15.93% in MDW to 24.87% in MDSW, and the ash content increased from 4.17% to 6.38%, indicating that deep fumigation promoted the formation of Maillard reaction products and the enrichment of minerals. The total sugar and pectin contents were highest in MSW (649.37 mg / g and 84.00 mg / g, respectively), and significantly decreased in MDSW (418.01 mg / g and 65.67 mg / g, respectively), indicating that deep fumigation intensified the thermal degradation of sugars and pectin or their consumption in the Maillard reaction. The protein content reached its highest level in MDSW (57.05 mg / g), significantly higher than that in MDW (42.39 mg / g) and MSW (25.65 mg / g), indicating that deep fumigation promoted the covalent binding of proteins and reducing sugars, allowing more protein components to be stably embedded in the melanin-like compound macromolecular structure. The free amino acid content was highest in MSW (32.67 mg / g) and slightly decreased to 26.01 mg / g in MDSW, reflecting that amino acids are activated as substrates in the early stages of the Maillard reaction and are continuously consumed through polymerization and cyclization in the advanced reaction stages. The total polyphenol and total flavonoid contents both peaked in MSW (57.44 mg / g and 48.11 mg / g, respectively) and significantly decreased in MDSW (38.70 mg / g and 33.37 mg / g, respectively), indicating that moderate smoking is beneficial for the retention of polyphenols and flavonoids, while excessive heating may lead to their oxidative degradation or irreversible complexation with melanoidins. Compared with ume melanoidins, MC exhibited extremely high protein content (119.67 mg / g) and total polyphenol content (72.80 mg / g), but extremely low pectin content (only 9.23 mg / g), reflecting the fundamental difference between coffee and ume raw material matrices.

[0051] 2. Molecular weight distribution The molecular weights of the four melanogens obtained in Example 1 were determined by gel permeation chromatography. The results showed that: MDW: average molecular weight 33.96±0.05 kDa; MSW: average molecular weight 50.68±0.10 kDa; MDSW: average molecular weight 163.92±0.11 kDa; MC: average molecular weight 52.03±0.44 kDa.

[0052] The results showed that the average molecular weight of melanin-like compounds in plums increased significantly with increasing fumigation intensity. Deep fumigation caused the molecular weight to increase dramatically to nearly five times that of MDW, indicating that heat fumigation significantly promoted the polymerization and cross-linking of melanin-like compounds. The molecular weight of MC was close to that of MSW, but much lower than that of MDSW.

[0053] 3. Zeta potential and particle size The zeta potentials of the four melanin species obtained in Example 1 (dispersed in 10 mM potassium phosphate buffer, pH=7) were measured using a Zetasizer Nano ZS, and the particle size distribution was determined by laser diffraction. The results showed that: MDW: zeta potential -26.23±0.85 mV, average particle size 703.967±34.419 nm; MSW: zeta potential -19.83±0.35 mV, average particle size 682.867±13.55 nm; MDSW: zeta potential -20.5±1.24 mV, average particle size 544.421±9.992 nm; MC: zeta potential -19.23±1.16 mV, average particle size 449.726±8.013 nm.

[0054] The results showed that all samples had a negative potential, with MDW exhibiting the highest absolute potential, indicating a strong electrostatic repulsion. With increasing fumigation intensity, the average particle size of the melanin-like polysaccharides gradually decreased, contrary to the increasing trend of molecular weight, suggesting that deep fumigation may have disrupted the physical entanglement of the polysaccharide chains, allowing for more complete dispersion of the particles in the aqueous phase.

[0055] 4. Water contact angle The water contact angles of the four types of melanin obtained in Example 1 were measured using an optical tensiometer. The results showed that: MDW had a water contact angle of 49.6±0.25°, indicating hydrophilicity; MSW had 51.3±0.5°, indicating amphiphilicity; MDSW had 87.3±0.2°, indicating a significant hydrophobic tendency; and MC had 41.9±0.61°, indicating strong hydrophilicity. The results indicate that with increasing fumigation intensity, the surface hydrophobicity of the melanin significantly increases, with MDSW's contact angle approaching 90°, while MC exhibits the strongest hydrophilicity. This trend is closely related to the hydrophobic structures such as aromatic rings and aliphatic side chains introduced during the Maillard reaction.

[0056] 5. Microscopic morphology Electron microscopy was performed on the four types of melanin obtained in Example 1, and their appearance characteristics were observed (see...). Figure 1 A) and electron microscopic observation (see A) Figure 1 See result B in the middle. Figure 1 As shown, the results are as follows. Figure 1 In the B category, the results showed that: MDW: loose and porous sheet-like structure with obvious wrinkles on the surface, large and unevenly distributed pores; MSW: more dense surface and more uniform structure; MDSW: uniform and dense honeycomb microporous structure with small and regularly distributed pores; MC: amorphous blocky aggregates with smooth surface and a small number of fine particles attached.

[0057] The results showed that MDW retained the original morphological characteristics of the polysaccharide backbone in the raw material; MDSW formed a uniform and dense honeycomb microporous structure, indicating that high-intensity thermal processing promoted the cross-linking and rearrangement between melanin molecules, forming a more ordered polymer structure; MC appeared as an amorphous block aggregate. These morphological differences are consistent with the chemical composition analysis, indicating that plum-type melanin has a polysaccharide backbone, while coffee-type melanin is mainly composed of protein-polyphenol complexes, and the two have fundamental differences in microstructure.

[0058] Example 3: Determination of emulsifying stability of melanin-like plum extracts 1. Preparation of emulsion samples Using the four types of melanin derived from dried plums (MDW, MSW, MDSW, and MC) obtained in Example 1 of this invention as emulsifying stabilizers, gelatin as a positive control, and an oil-water mixture without stabilizers as a blank control, an oil-in-water emulsion was prepared. The specific operation is as follows: Weigh out 2% by mass of various melanin powders and disperse them in deionized water. Stir magnetically at 4°C for 12 hours to fully hydrate the melanin-like substances and obtain an aqueous phase. Use corn oil as the oil phase, accounting for 15% of the total system mass. After mixing the aqueous and oil phases evenly, homogenize them using a high-pressure homogenizer at 13500 r / min for 5 minutes to obtain a homogeneous and stable oil-in-water emulsion. The blank control group is mixed with only corn oil and deionized water, without adding any emulsifying stabilizers.

[0059] All prepared emulsions were stored at 20°C in the dark for 21 days. Samples were taken at regular intervals on storage days 0, 3, 5, 7, 14, and 21 to conduct macroscopic appearance observation, microscopic morphology observation, droplet size distribution detection, and rheological property characterization.

[0060] 2. Macroscopic appearance and microscopic morphology characterization of emulsions During the storage period, the macroscopic layering, water separation, and fading of the emulsion were observed with the naked eye, and the flocculation and aggregation behavior of oil droplets were observed using an optical microscope. The results are as follows: Figure 2 As shown.

[0061] The blank control group (emulsion without stabilizer) had extremely poor stability. Significant oil-water phase separation appeared by day 5 of storage, and complete stratification occurred by day 7, with the oil phase floating on the upper layer. Microscopic observation showed that the oil droplets in the fresh emulsion were unevenly distributed in size. After 5 days of storage, the oil droplets flocculated and aggregated, forming large oil droplets of 10-40 μm. After 7 days of storage, the continuous oil phase dominated the field of view, and the emulsion system was completely demulsified. The reason is that there is no stable interfacial film at the oil-water interface, which cannot prevent oil droplet collision and aggregation.

[0062] The gelatin-stabilized emulsion (positive control) was initially uniform, but water separation appeared at the bottom starting from the 7th day of storage. The amount of water separation continued to increase with the extension of storage time. After 21 days of storage, a large number of oil droplets were observed to flocculate and aggregate to form agglomerates. Although gelatin can initially form a protein film at the oil-water interface, the mechanical strength of the interfacial film is limited. Long-term storage can easily cause the film to rupture, leading to irreversible aggregation of oil droplets.

[0063] Slight water separation occurred in the MC stable emulsion on the 7th day of storage, and the total amount of water separation on the 21st day was higher than that of the four ume-derived melanin experimental groups. Under the microscope, the oil droplets slowly increased in size with storage, with only slight aggregation and no large-scale aggregation. MC is a coffee-derived melanin with strong intermolecular association of protein-polyphenol complex, but the interfacial spreading rate is weaker than that of ume-derived melanin, and the interfacial elasticity is lower. Therefore, its storage stability is not as good as that of MDW, MSW, and MDSW.

[0064] Slight phase separation occurred in MDW stabilized emulsion on day 5 of storage, and water separation and fading were significantly aggravated after day 7. Oil droplet flocculation could be observed under a microscope after day 5, and the degree of aggregation continued to increase after day 7, generating a large number of large-diameter oil droplets. MDW had a shallow degree of drying, a low degree of Maillard reaction, an insufficient number of amphiphilic functional groups, and a weak adsorption capacity at the oil-water interface, making it difficult to construct a dense and complete interfacial protective film.

[0065] MDSW stabilized emulsion exhibits excellent overall uniformity, with only a trace amount of bottom water separation observed after 14 days of storage; the oil droplet size distribution is uniform throughout the process, with only a very slight oil droplet aggregation observed after 14 days; MDSW prepared by deep fumigation has a high degree of polymerization, which can form a dense adsorption layer at the interface to block oil droplet collisions, but the excessively strong intermolecular forces will slow down the interface spreading speed, and the overall storage stability is slightly lower than that of MSW.

[0066] MSW-stabilized emulsion exhibited the best stability in this embodiment, maintaining a uniform milky white appearance throughout the 21-day storage period. Only trace amounts of water were observed at the bottom, with no stratification, significant flocculation, or oil droplet aggregation. Under a microscope, the oil droplets were uniformly dispersed throughout the process, with no significant increase in particle size, and no flocculation or aggregation behavior was observed. This is attributed to the moderate Maillard reaction of MSW, whose molecules are rich in both hydrophilic groups such as phenolic hydroxyl and carboxyl groups and aromatic hydrophobic segments, resulting in optimal amphiphilic balance. This allows for rapid adsorption at the oil-water interface to construct a dense, highly elastic interfacial film, inhibiting oil droplet collisions through a combination of steric hindrance and electrostatic repulsion. Simultaneously, moderate polymerization increases the viscosity of the continuous phase, slowing the oil droplet migration rate and maintaining emulsion stability over a long period.

[0067] 3. Characteristics of emulsion droplet size distribution Laser diffraction was used to detect the droplet size distribution and average droplet diameter of emulsions stored for 0 days, 3 days, 5 days, 7 days, 14 days, and 21 days, respectively. The droplet size distribution curves and average droplet diameter statistical results are shown below. Figure 3 As shown.

[0068] All freshly prepared (0d) emulsions exhibited a narrow single-peak particle size distribution, with oil droplet sizes less than 5μm, indicating that high-pressure homogenization could achieve uniform dispersion of the oil phase. With prolonged storage, the evolution of particle size distribution varied significantly among the groups: the blank control group showed obvious aggregation after 3 days of storage, with the main peak shifting to 10-100μm and the appearance of secondary large-diameter peaks; the average particle size increased sharply after 7 days, and the emulsion became completely unstable; the gelatin-stabilized emulsion showed large-diameter shoulder peaks from 3 days onwards, with the average particle size continuously increasing; the particle size distribution broadened significantly after 21 days, and its stability was weaker than all melanoidin-like experimental groups.

[0069] Comparing the four types of melanin emulsions, MDW was stable for only 7 days, after which the curve showed a large-diameter shoulder peak, indicating intensified oil droplet flocculation and a significant increase in average particle size; MDSW was stable for 14 days, with only a slight increase in particle size at 21 days; MSW maintained an excellent single-peak distribution throughout the complete 21-day storage period, with almost no large-diameter oil droplet formation, and only a slight broadening of the distribution at 21 days, exhibiting the smallest change in average particle size and the best anti-agglomeration ability; MC showed a slow shift in particle size towards larger sizes throughout the storage process, with moderate agglomeration, and an average particle size increase higher than the three types of melanin-like plums but lower than the gelatin control group.

[0070] The average particle size bar chart provides a clear and quantitative ranking of the emulsion storage stability: MSW > MDSW > MC > MDW > Gelatin > Blank. Droplet size variation directly reflects the interfacial adsorption tightness and anti-agglomeration ability of the emulsion stabilizers. The blank group lacks a protective interfacial film, leading to rapid droplet aggregation. Gelatin's interfacial film has insufficient mechanical strength. MDW has a low degree of polymerization and a lack of active functional groups, making it prone to bridging and flocculation. MC's interfacial spreading and film strength are weaker than those of melanin-based emulsions. MSW's moderate Maillard reaction results in a balanced amphiphilic structure, enabling rapid film formation and relying on steric hindrance and electrostatic repulsion to prevent oil droplet contact. Simultaneously, moderate polymerization increases the viscosity of the continuous phase, maximally inhibiting oil droplet migration and aggregation. MDSW's highly polymerized molecules construct a dense interfacial layer, exhibiting strong anti-collision ability, but its molecular spreading rate is limited, resulting in slightly lower stability than MSW.

[0071] 4. Characterization of emulsion rheological properties Steady-state shear tests, frequency sweep tests, and strain sweep tests were conducted on the blank group, gelatin control group, and four types of melanoidin-stabilized emulsions using a rotational rheometer. The frequency sweep range was 0.1–10 rad / s, and the strain was fixed at 1%. The rheological curves are shown below. Figure 4 As shown.

[0072] Steady-state shear test ( Figure 4As shown in A), all emulsions exhibited pseudoplastic fluid characteristics with shear thinning, and the apparent viscosity decreased rapidly with increasing shear rate. The blank emulsion had the lowest apparent viscosity, while the gelatin emulsion had a higher viscosity than the blank but lower viscosity than the ume-based melanin group. Among the ume-based melanin groups, MSW and MDSW had significantly higher apparent viscosities than MDW and MC, indicating that they constructed a denser micro-network structure in the continuous phase of the emulsion. The viscosity of MC was intermediate, higher than that of gelatin but lower than that of MSW / MDSW.

[0073] Frequency scan results ( Figure 4 (B~E) indicates that the storage modulus G′ of all emulsions is greater than the loss modulus G″, and the system as a whole exhibits an elastic-dominated gel-like three-dimensional network structure. MSW and MDSW have the highest storage modulus G′ values, which increase significantly with increasing angular frequency, indicating that the gel network structures formed by these two groups are the most robust. MDW and gelatin have lower storage moduli. Complex viscosity η *The variation pattern matches the storage modulus. MSW and MDSW have the highest complex viscosity, which increases continuously with frequency, a typical characteristic of strong gel systems. The loss factor tanδ (G″ / G′) can characterize the viscoelastic balance of the system. MSW and MDSW have the lowest tanδ values ​​and flat curves, indicating stable internal network structure and excellent resistance to deformation. Gelatin and the blank group have higher tanδ values, indicating weak system elasticity, easy structural damage, and poor storage stability.

[0074] Strain scanning test ( Figure 4 The F~I values ​​in the equation are used to determine the linear viscoelastic range and the critical strain for structural failure of the emulsion: In the low strain range (<1%), G′ remains constant for all groups, indicating that they are in the linear viscoelastic range; MSW and MDSW have the widest linear viscoelastic range, the largest critical strain for network structure fracture, and the best resistance to shear failure; Under high strain conditions, MSW and MDSW have the slowest tanδ rise rate and remain at a low level overall, further confirming that they can form a highly elastic and stable gel-like continuous phase network.

[0075] 5. Comprehensive performance analysis and application prospects Based on the combined characterization results of microstructure, droplet size, and rheological properties, the emulsification stability of the four types of plum-derived melanin was ranked as MSW > MDSW > MDW, all of which were superior to coffee-derived melanin MC and the traditional emulsification stabilizer gelatin. Among them, MSW was the optimal emulsification stabilizer of this invention.

[0076] MSW combines a balanced amphiphilic molecular structure with a moderate degree of polymerization. On one hand, it can rapidly adsorb at the oil-water interface to form a highly elastic and dense protective film, relying on a dual mechanism of electrostatic repulsion and steric hindrance to prevent oil droplet flocculation. On the other hand, it can construct a highly elastic three-dimensional gel network in the aqueous continuous phase, increasing the system viscosity and restricting oil droplet migration and floating, maintaining the emulsion's homogeneity and stability for a long period of 21 days in the dark at room temperature. MDSW has excellent interfacial barrier effects due to its high polymerization, but its molecular spreading rate is limited; MDW has insufficient Maillard reaction and lacks amphiphilic groups, resulting in weaker long-term stability.

[0077] Compared to animal-derived emulsifiers such as gelatin, the plum-derived melanin of this invention is a plant-based natural product that has multiple activities including emulsification stability, anti-oxidation, and inhibition of digestive enzymes, and has no risk of animal-derived allergies. Compared to coffee-derived melanin (MC), plum-derived melanin forms a film faster at the interface, has a stronger viscoelastic network, and has significantly improved storage stability.

[0078] The melanin derived from plum in this invention can be used as a green, multifunctional emulsifying stabilizer in plant-based beverages, emulsion-type functional diets, nano-delivery carriers, cosmetic emulsions, and other systems. While achieving long-lasting emulsion stability, it simultaneously imparts additional physiological activities such as antioxidant and postprandial blood glucose regulation to the products, and has broad application value in the food and daily chemical industries.

[0079] Example 4: Determination of Colon-Targeted Delivery Performance of Deeply Smoked Prunus Melanin-like Extract (MDSW) 1. In vitro simulated digestion experiment A static three-step in vitro digestion model was used to simulate the digestive environment of the human stomach, small intestine, and colon. The encapsulation, protection, fractional controlled release, and colon-targeted delivery capabilities of four melanin species (MDW, MSW, MDSW, and MC) as polyphenol carriers were evaluated. The preparation methods for each digestive solution are as follows: Simulated gastric juice (SGF, pH=1.2): 30 mg of pepsin was added to deionized water, and the pH of the system was adjusted to 1.2 using 1.0 mol / L hydrochloric acid to create a strongly acidic digestive environment in the stomach; Simulated intestinal fluid (SIF, pH=6.8): 100 mg of porcine bile salts and 30 mg of pancreatic enzymes were added to deionized water, and the pH of the system was adjusted to 6.8 using 7.0 mol / L sodium hydroxide to construct a weakly acidic digestive environment in the small intestine; Simulated colonic fluid (SCF, pH=7.4): β-mannanase was added to deionized water, and the pH of the system was adjusted to 7.4 using 7.0 mol / L sodium hydroxide to construct a weakly alkaline digestive environment in the colon.

[0080] Polyphenol-loaded samples (MDW, MSW, MDSW, and MC) were prepared, with free polyphenol solution serving as a control. Each sample was dispersed in simulated gastric fluid and incubated at 37°C with stirring for 3 hours, with polyphenol release measured every hour. After gastric digestion, the entire system was transferred to simulated intestinal fluid and incubated for 3 hours, with samples measured every hour. Finally, the system was transferred to simulated colonic fluid and incubated for 6 hours, with cumulative polyphenol release rate measured throughout the process. The cumulative polyphenol release results for each group are shown below. Figure 5 .

[0081] 2. Analysis of the segmented release pattern and conclusions of polyphenols Depend on Figure 5 As shown in the cumulative polyphenol release curves, the polyphenol release behavior of the four types of carriers exhibits significant segmental differences. The release characteristics are directly related to the carrier molecular polymerization structure and the acid-base environment of the digestive tract. The segmental results are as follows: (1) Stomach digestion stage (0~180 min, pH=1.2 strong acid environment): The release rate of polyphenols from coffee-derived melanin MC was the fastest, with a cumulative release rate of 78.68±5.25% after 3 hours of incubation. MC binds polyphenols through hydrogen bonds and hydrophobic interactions. Under acidic conditions, the intermolecular forces are destroyed, and the polyphenols dissociate and dissolve rapidly. The sustained-release effect of ume-derived melanin is significantly better than that of MC. Among them, MDSW has the lowest release rate, with a release rate of only 41.65±2.11% after 180 min, which is significantly lower than that of MDW (49.59±4.56%) and MSW (52.65±3.74%). The deep smoking process enables MDSW to form a high-polymerization, dense macromolecular network. The polyphenols are stably encapsulated inside the molecular skeleton. Under strong acid conditions, the carrier structure is not easily disintegrated, which can effectively inhibit the premature release of polyphenols and achieve acid resistance protection in the stomach.

[0082] (2) Small intestine digestion stage (180~360min, pH=6.8 weak acid environment): The release rate of polyphenols in all samples was greatly increased. The weak alkaline environment promoted the swelling and local degradation of the melanoidin network, which encapsulated the polyphenols and released them outward continuously. Among them, MDSW showed the most significant increase in release, with a cumulative release rate of 82.55±4.56% at 360min, and a large amount of polyphenols were released in a controllable manner. MC, on the other hand, showed a slight increase in release during this stage, with most of the polyphenols already released in the stomach in advance.

[0083] (3) Colonic digestion stage (360~600min, pH=7.4 weakly alkaline environment): The polyphenol release rate of each group gradually tends to be slow. At the end of digestion at 600min, the cumulative release rate of polyphenols in all samples exceeded 95%, and polyphenols could be completely released.

[0084] Based on the overall release pattern throughout the entire cycle, we can see that: MC and MDW are fast-release carriers in the stomach, and polyphenols are easily oxidized and degraded by gastric acid and pepsin, resulting in severe loss of activity; MSW has a certain sustained-release capacity, but its targeted enrichment effect is limited; MDSW exhibits typical delayed-release characteristics, with polyphenols mainly released in the small intestine and colon, which can avoid the destruction of polyphenol activity by strong gastric acid and has excellent colon-targeted delivery capability.

[0085] 3. Mechanism of Action and Application Effect Analysis (1) Targeted delivery mechanism The colon-targeted controlled-release performance of MDSW is determined by its highly polymerized and dense network structure induced by deep smoking: in the highly acidic environment of the stomach, the highly cross-linked macromolecular backbone structure is stable, and the closed pores lock in polyphenols, achieving "lock-in release" in the stomach; after entering the weakly alkaline system of the small intestine and colon, the carrier molecules swell, and at the same time, β-mannanase locally degrades the backbone, gradually opening the network pores, and releasing polyphenols continuously and stably to the colon. In contrast, MDW has a shallow degree of drying, a low degree of Maillard reaction, a loose molecular structure, and poor encapsulation stability; ordinary smoked MSW has a moderate degree of polymerization, but its density is not as good as MDSW, and its gastric lock-in effect is weak; coffee-derived MC relies on non-covalent weak interactions to bind polyphenols, has poor acid and alkali tolerance, and cannot achieve intestinal targeted delivery.

[0086] (2) Industrial application value The colon is a core site for anti-inflammatory, gut microbiota regulation, and metabolic control in the human gut. The antioxidant, anti-inflammatory, and gut microbiota-regulating effects of polyphenols require accumulation in the colon to be fully realized. Free polyphenols or polyphenols encapsulated in MC or MDW carriers are released in large quantities in the stomach, but are easily destroyed by gastric acid and have difficulty reaching the colon. In contrast, the MDSW of this invention can protect polyphenols and allow them to pass through the stomach intact, delivering the active substances to the colon for complete release and maximizing the preservation of polyphenol physiological activity.

[0087] The MDSW of this invention is a natural plant-based carrier with no risk of animal-derived allergies. It can be used to prepare products such as colon-targeted functional diets, intestinal sustained-release oral liquids, and oral polyphenol nanodelivery formulations. At the same time, MDSW itself has antioxidant and digestive enzyme activity inhibition properties. The carrier and active polyphenols have synergistic effects, and it has broad application prospects in the fields of functional foods and oral intestinal conditioning preparations.

[0088] Example 5: Verification of the in vitro antioxidant and hypoglycemic activities of melanin-like substances from dried plums 1. In vitro antioxidant activity assay (DPPH, ABTS, FRAP reducing power method, corresponding to...) Figure 6 AC in Using MDW, MSW, MDSW, and MC as test samples, and ascorbic acid and water-soluble vitamin E as positive controls for antioxidant activity, a series of concentration gradient solutions were prepared. The in vitro antioxidant capacity of each sample was simultaneously evaluated using DPPH free radical scavenging assay, ABTS free radical scavenging assay, and FRAP total reducing power assay. Detailed concentration-activity curves for each group are shown below. Figure 6 A, B, and C in the diagram.

[0089] (1) Experimental procedure and results of DPPH free radical scavenging The sample solutions of varying concentrations were thoroughly mixed with DPPH ethanol solution, and allowed to stand in the dark for 30 minutes. The absorbance of the system was measured at a wavelength of 517 nm, and the free radical half-scavenging concentration (IC50) of each sample was calculated. 50 Positive control: ascorbic acid IC 50 =56.32±3.25 μg / mL; IC50 of MC in melanoidin samples 50 =0.62±0.05 mg / mL, IC50 of MSW 50 =1.25±0.08mg / mL, MDWI C 50 =1.87±0.12 mg / mL, IC50 of MDSW 50 =2.34±0.15 mg / mL. The order of DPPH free radical scavenging activity of each sample from strongest to weakest is: MC>MSW>MDW>MDSW.

[0090] Coffee-derived MC protein-polyphenol complex structure is rich in free electron-donating groups, which can quickly capture free radicals and has the best antioxidant activity; the MSW in ume-derived melanin has a moderate degree of Maillard reaction, rich content of intramolecular reducing ketones and phenolic active intermediates, and sufficient exposure of active sites, and its antioxidant performance is better than MDW and MDSW; MDW has a shallow degree of drying and processing, and the total amount of active products generated by Maillard reaction is relatively low; the deeply smoked MDSW molecules are highly cross-linked and polymerized, and the active groups are wrapped by a macromolecular network. The steric hindrance hinders the transfer of hydrogen atoms, and its free radical scavenging ability is the weakest.

[0091] (2) Verification of ABTS radical scavenging and FRAP total reducing power ABTS free radical scavenging curve ( Figure 6 (B) FRAP total reduction absorbance curve ( Figure 6 The trend of C) in the experiment was completely consistent with that of the DPPH experiment. Both showed a dose-dependent characteristic of activity gradually saturating with increasing concentration, which once again confirmed that MC had the best antioxidant capacity. MSW was the sample with the best antioxidant performance among the three types of melanin in plum in this invention. The reduction electron-donating ability of overpolymerized MDSW decreased significantly.

[0092] 2. In vitro assay of the inhibitory activity of hypoglycemic digestive enzymes (α-glucosidase and α-amylase inhibition methods, corresponding to...) Figure 6 DE in Using acarbose as a positive control for hypoglycemia, the inhibitory effects of MDW, MSW, MDSW, and MC on the key starch digestion enzymes α-glucosidase and α-amylase were determined. The enzyme inhibition rate versus concentration curves are detailed below. Figure 6 D and E in the text.

[0093] (1) Results of α-glucosidase inhibition experiment The sample solutions of varying concentrations were premixed with α-glucosidase buffer and incubated. The substrate 4-nitrophenyl-α-D-glucopyranoside was then added, and the reaction was carried out at 37°C for 20 min. The absorbance was measured at 405 nm, and the half-inhibition concentration (IC50) was calculated. 50 Positive control acarbose IC 50 =10.5±1.02 μg / mL; IC50 of the melanoidin-like MDW to be tested 50 =1.62±0.10 mg / mL, IC50 of MSW 50 =2.15±0.12 mg / mL, IC50 of MC 50 =2.52±0.15 mg / mL, IC50 of MDSW 50 =2.86±0.18 mg / mL. The α-glucosidase inhibitory activity of each sample was ranked as follows: MDW>MSW>MC>MDSW.

[0094] (2) α-Amylase Inhibition Experiment and Comprehensive Conclusion α-Amylase inhibition experiment ( Figure 6 The order of activity strength of E in the samples was consistent with that of α-glucosidase, and all samples showed higher inhibition efficiency against α-glucosidase than against α-amylase.

[0095] Mechanism analysis: Shallow-dried MDW has a low degree of Maillard polymerization, with a large number of uncondensed sugar groups and phenolic hydroxyl groups exposed within the molecule. These can be stably bound to the active site of enzymes through hydrogen bonding and hydrophobic interactions, thus blocking the substrate catalytic process. As the smoking processing depth increases, the degree of polymerization and cross-linking of MSW and MDSW molecules gradually increases, and the active sites are shielded by the macromolecular backbone, resulting in a continuous weakening of their binding ability to digestive enzymes. Although coffee-derived MC is rich in polyphenols, the protein-polyphenol complex brings steric hindrance, making its enzyme inhibitory effect weaker than that of MDW and MSW.

[0096] 3. Comprehensive Activity and Application Effect Analysis Based on the combined results of in vitro antioxidant and carbohydrate digestion enzyme inhibition activities, the four types of melanoidins in this invention possess differentiated functional advantages: MC exhibits the best free radical scavenging and reducing antioxidant properties, making it suitable as a natural antioxidant additive; MSW combines excellent antioxidant properties with moderate hypoglycemic enzyme inhibition activity, making it a multifunctional compound raw material; MDW has the strongest inhibitory ability against α-glucosidase and α-amylase, making it preferentially suitable for functional foods that regulate postprandial blood glucose; Deeply smoked MDSW has relatively weak antioxidant and hypoglycemic enzyme inhibition activities, but based on the excellent emulsification stability and colon-targeted delivery performance demonstrated in the previous examples, it can be used as a polyphenol colon-release carrier.

[0097] Compared to chemically synthesized antioxidants and hypoglycemic agents, the melanin derived from ume in this invention is a natural Maillard reaction product with high safety and no chemical additive residues. It can be used alone or in combination in products such as meal replacements, plant-based milk beverages, baked goods, and intestinal-targeted dietary supplements, simultaneously achieving multiple effects such as antioxidant preservation, stabilizing postprandial blood sugar, and slow release of active substances into the intestines.

[0098] Example 6: Stability Verification of Prunus mume-like Melanin 1. Indoor long-term sunlight storage stability test Aqueous solutions of MDW, MSW, MDSW, and MC melanoidins with a concentration of 0.5 mg / mL were prepared and stored in a sealed container under natural light at room temperature for 54 days. The absorbance changes of the characteristic absorption peak of the conjugated chromophore at 280 nm were continuously monitored using a UV spectrophotometer. The absorbance of the sample on storage day 0 was used as the 100% baseline to calculate the melanoidin degradation rate at different storage durations. The degradation curves are shown below. Figure 7 A in the middle.

[0099] During the storage period, the absorbance of all samples continuously decreased with prolonged storage time, indicating only slow photo-oxidative degradation. No new secondary absorption peaks were observed in the UV spectra. The degradation rates of the samples after 54 days of sunlight storage were ranked as follows: MDW > MDSW > MSW > MC. MDW exhibited a shallow Maillard reaction and a high number of uncondensed glycosidic and peptide bonds within its molecules, making it prone to oxidative hydrolysis under light and resulting in the fastest degradation rate. MSW and MDSW, after gradient fumigation and Maillard polymerization, formed dense macromolecular networks with superior conjugated backbone stability compared to MDW. Among them, moderately fumigated MSW showed a moderate degree of crosslinking and exhibited better sunlight stability than deeply fumigated MDSW.

[0100] 2. Accelerated Storage Stability Test under 365nm Ultraviolet Irradiation Each group of 0.5 mg / mL melanoidin aqueous solution was continuously irradiated under a 6W, 365 nm UV lamp for 15 days. The absorbance at 280 nm was monitored simultaneously, and the degradation rate was calculated. The degradation curves are detailed below. Figure 7In section B, ultraviolet (UV) irradiation significantly accelerates the degradation of melanoidins. The degradation rate after 15 days of UV irradiation is close to or even exceeds that of samples stored under indoor sunlight for 54 days. Some samples exhibit shoulder peaks in their UV spectra within the 260-270 nm range, confirming that UV light directly excites the conjugated aromatic structures of molecules, initiating complex fragmentation of the macromolecular skeleton. The UV degradation rates of the samples are ranked as follows: MDSW > MC > MDW > MSW. Deeply smoked MDSW exhibits the highest molecular conjugation and aromatic ring density, strong UV absorption, and the most significant UV-induced oxidative fragmentation. MSW has a moderate conjugated structure and the best UV tolerance. MC has a high content of polyphenol aromatic groups, making it easily oxidized and decomposed under UV irradiation, resulting in a significant decrease in UV stability.

[0101] 3. Stability test under different pH and acid-base environments The pH of the four types of melanoidin aqueous solutions was adjusted to 1.0, 3.0, 5.0, 7.0, 9.0, 11.0, and 13.0, respectively. A sample with the original natural pH (approximately 6.5) was used as a control. After being sealed and stored at room temperature for 7 days, the absorbance at 280 nm was measured, and the degradation rate of each group was calculated. A bar chart comparing the degradation rates at different pH values ​​is shown below. Figure 7 C in the middle.

[0102] Acidic and alkaline environments can alter the dissociation state of melanoidin functional groups and molecular conformation, significantly affecting the stability of the molecular skeleton. (1) The most severe degradation range for each group of samples was in a strongly acidic environment (pH=1.0). The degradation rates were 45.2±3.5% for MDW, 32.5±2.8% for MSW, 28.6±2.5% for MDSW, and 18.5±2.2% for MC. Under strong acid conditions, the carboxyl and phenolic hydroxyl groups are protonated, and the intramolecular hydrogen bonding is enhanced. At the same time, acid catalyzes the hydrolysis of glycosidic bonds and peptide bonds, destroying the conjugated color structure. MDW has the lowest Maillard polymerization degree, the largest number of uncondensed chemical bonds, and the worst tolerance to acid hydrolysis.

[0103] (2) In the neutral to weakly alkaline range (pH=7.0~9.0), the degradation rate of all samples was only 5%~15%, which is the optimal range for stability. In this pH range, the molecular functional groups are moderately dissociated and the molecular chains are extended. There is neither strong acid catalytic hydrolysis nor strong base oxidative cracking. The high polymer network of MDSW after deep smoking further enhances the tolerance to neutral environment.

[0104] (3) The degradation rate increased significantly again in a strongly alkaline environment (pH=13.0). The degradation rate of melanin from ume was concentrated in 25%~30%, while the degradation rate of MC from coffee rose to 35.2±3.0%. Under strongly alkaline conditions, the functional groups were completely deprotonated, and the repulsion of the negative charge of the molecule caused the chain to extend. At the same time, the strongly alkaline oxidation system destroyed the conjugated double bond and aromatic ring structure. The MC polyphenol component was easily oxidized in strongly alkaline conditions, and the stability reduction was greater than that of melanin from ume.

[0105] Overall acid-base stability pattern: MC has the best stability across the entire pH range, while MDW has the worst acid resistance; MDSW has outstanding stability in neutral environments, while MSW has a balanced overall acid and base tolerance.

[0106] Based on the results of stability tests under conventional storage, ultraviolet irradiation, and a wide range of pH conditions, the physicochemical stability of the three types of melanin from dried plums showed significant differences: MDW had the weakest tolerance to sunlight and acids / alkalis, and was only suitable for short-shelf-life systems stored in the dark and at near-neutral pH; MSW showed balanced performance under sunlight, ultraviolet irradiation, and acid / alkali conditions, and was suitable for most conventional food emulsions and functional beverage systems; MDSW had excellent stability in a neutral environment, but was easily degraded under ultraviolet light, and was suitable for neutral enteric delivery formulations stored in the dark.

[0107] The results of emulsification performance, colon-targeting, and in vitro activity tests in the aforementioned embodiments confirm that the present invention can directionally prepare a series of melanin-like substances with controllable functions by regulating the drying and smoking gradient thermal processing process; moderately smoked MSW exhibits outstanding emulsification stability, and its long-lasting emulsion stability is superior to that of gelatin and coffee-based melanin, making it suitable as a multifunctional natural emulsifying stabilizer; deeply smoked MDSW possesses a unique colon-targeting controlled-release effect; MDW exhibits the strongest inhibitory activity against α-glucosidase and α-amylase; and it demonstrates excellent antioxidant and long-term storage stability.

[0108] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for the directional preparation of melanin from dried plums with adjustable functions, characterized in that, Includes the following steps: (1) The dried plum fruit was dried by hot air to obtain the dried plum precursor; (2) The ume precursor is subjected to fumigation treatment. By controlling the fumigation temperature and time, a Maillard reaction gradient is constructed to obtain ume raw materials with different degrees of Maillard reaction. (3) The raw materials of different Maillard reaction degrees obtained in step (2) were defatted, ultrasonically assisted to extract, solid-liquid separated, concentrated, alcohol precipitated, ultrafiltration purified and dried to obtain functionally differentiated melanin of plum. Among them, the functionally differentiated melanin of plum includes at least emulsified stable melanin of plum and colon-targeted sustained-release melanin of plum.

2. The directional preparation method according to claim 1, characterized in that, The hot air drying conditions described in step (1) are: temperature 80 ℃, wind speed 1.5 m / s, time 72 h, drying until the moisture content is ≤5%.

3. The directional preparation method according to claim 1, characterized in that, The Maillard reaction gradient mentioned in step (2) includes: Conventional fumigation gradient: fumigation temperature is 60~80 ℃, fumigation time is 72 h, to obtain plum raw material with moderate Maillard reaction degree, which is used to prepare emulsion-stabilized plum melanin; Deep fumigation gradient: The fumigation temperature is 80~110 ℃ and the fumigation time is 96 h to obtain plum raw materials with a high degree of Maillard reaction, which are used to prepare colon-targeted sustained-release plum melanin.

4. The directional preparation method according to claim 1, characterized in that, The conditions for ultrasonic-assisted extraction in step (3) are as follows: the extraction solvent is an ethanol aqueous solution with a volume concentration of 8%~12%, the material-liquid ratio is 1:15~1:25 g:mL, the extraction temperature is 75~85 ℃, the extraction time is 25~35 min, the ultrasonic power is 250~350 W, and the frequency is 35~45 kHz.

5. The directional preparation method according to claim 1, characterized in that, The alcohol precipitation in step (3) is as follows: add anhydrous ethanol to the concentrate to a final concentration of 75%~85%, let stand for 10~14 h, and then centrifuge to collect the supernatant; the ultrafiltration purification is carried out by 2~4 cycles of ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa.

6. The application of the directional preparation method according to any one of claims 1 to 5 in the preparation of functionally adjustable melanin-like plum extract.

7. The melanin-like substance of dried plum prepared by the directional preparation method according to any one of claims 1 to 5, characterized in that, The melanin-like substance from dried plums has the following characteristics: (i) The total polysaccharide content is 418.01~649.37 mg / g, the pectin content is 65.67~84.00 mg / g, and the protein content is 25.65~57.05 mg / g; (ii) The average molecular weight distribution range is 33.96 × 10⁻⁶. 3 Da~163.92×10 3 Da; (iii) As the smoking intensity increases, its microstructure gradually evolves from a loose and porous sheet-like structure to a uniform and dense honeycomb microporous structure, and the water contact angle increases from 49.6° to 87.3°.

8. The melanin-like substance derived from plum according to claim 7, characterized in that, The melanin-like substances from dried plums include: Emulsified and stabilized plum melanin, prepared by the conventional fumigation gradient, has a water contact angle of 51.3±0.5°. It can adsorb at the oil-water interface to form a dense and elastic interfacial film and form a three-dimensional gel network in the continuous phase. The colon-targeted sustained-release melanin-like substance, prepared by the aforementioned deep fumigation gradient, has a water contact angle of 87.3±0.2°. Its polyphenol release rate in simulated gastric juice is only 41.65%, while its polyphenol release rate in simulated intestinal juice and simulated colonic juice can reach over 95%.

9. The melanin-like substance derived from plum according to claim 7 or 8, characterized in that, The DPPH free radical scavenging IC of the melanin-like substance of ume 50 Values ​​range from 1420 to 1762 μg / mL, with an IC50 value for ABTS free radical scavenging. 50 The values ​​ranged from 571.56 to 830.25 μg / mL, and the IC50 value for α-glucosidase inhibition was [value missing]. 50 The value ranged from 49.5 to 72.56 μg / mL, and the IC50 value for α-amylase inhibition was 49.5–72.56 μg / mL. 50 The values ​​ranged from 38.5 to 60.5 μg / mL.

10. The use of the plum-type melanin as described in any one of claims 7 to 9 in food emulsions or colon-targeted delivery systems.

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